Retinitis pigmentosa (RP) is an inherited degenerative disease of the retina that affects approximately one in 3,500 individuals, with an estimated 1.5 million patients worldwide. See Churchill et al., 2013, Invest. Ophthalmol. Vis. Sci. 54(2): 1411-1416. RP is caused by progressive loss of rod and cone photoreceptors, resulting in night blindness followed by loss of visual fields. The disease may result in legal or even complete blindness. Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for greater than 70% of the cases of human X-linked retinitis pigmentosa (XLRP), the most severe subtype of RP. See Beltran et al., 2012, PNAS 109(6): 2132-2137 and Bader et al., 2003, Invest. Ophthalmol. Vis. Sci. (44)4: 1458-1463.
Alternative splicing of the RPGR gene results in expression of multiple isoforms of the RPGR protein. The mRNA for isoform A contains all 19 exons of the gene, while the mRNA for isoform C contains exons 1 to 15 and a large part of intron 15. Intron 15 is a purine-rich region that contains highly repetitive sequences that code for glutamate and glycine repeats (EEEGEGEGE in human (SEQ ID NO: 9) and EEGEGE in mouse (SEQ ID NO: 10)), see Vervoort et al., Mutational hot spot within a new RPGR exon in X-linked retinitis pigmentosa. Nat Genet 2000; 25:462-6. Isoform A is constitutively expressed in all tissues while isoform C, which is also referred to as “ORF15”, is the predominant form expressed in the connecting cilium of photoreceptor, see Hong et al., Invest Ophthalmol Vis Sci 2002; 43:3373-82, and Hong et al., Invest Ophthalmol Vis Sci 2003; 44:2413-21.
A total of 55% of RPGR-related XLRP is caused by mutations in ORF15, all of which result from deletions that lead to truncated proteins. Most of the other cases are caused by mutations in exons 1-13, which can be either missense or nonsense mutations, with a small number caused by mutations in introns or large deletions. No cases have been identified due to mutations in exons 16 to 19.
Recent studies have demonstrated the potential of gene therapy approaches to treating XLRP caused by mutations in the RPGR gene. For example, Beltran et al. have shown that subretinal injections of adeno-associated virus (AAV) vectors expressing human RPGR increased rod and cone photoreceptor function in a canine model of XLRP.
However one of the challenges in large-scale production of AAV vectors for clinical use is that nucleic acid sequences encoding a protein of interest such as RPGR may be unstable, resulting in the accumulation of several mutations and deletions. For example, the RPGR gene contains a region of 1.2 kb called ORF15 near the 3′ end of the cDNA that is highly repetitive and GA rich. This region is a mutation “hot spot” in population. This repetitive region is very unstable during cloning and vector preparation and clones obtained generally contain mutations and deletions. These mutations can potentially alter or eliminate RPGR protein function, limiting the use of this protein in gene therapy applications. Therefore a need exists to identify methods of stabilizing RPGR cDNAs during large-scale production of AAV vectors.